Substituted-hydroxy-naphthalenedisulfonic acid compounds

ABSTRACT

Substituted-hydroxy-naphthalenedisulfonic acids, acid ureides and salts thereof useful as complement inhibitors.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to concurrently filed and copendingapplication Ser. No. 684,695, now U.S. Pat. No. 4,018,764.

BACKGROUND OF THE INVENTION

The present invention resides in the concept of certain(substituted)hydroxy-naphthalenedisulfonic acids, acid ureides and saltsthereof, particularly new (substituted)4-hydroxy-2,7-naphthalenedisulfonic acids and known (substituted)4-hydroxy-2,7-naphthalenedisulfonic acids and4-hydroxy-2,7-naphthalenedisulfonic acid ureides, all of which areuseful as inhibitors of the complement system of warm-blooded animals.

The term "complement" refers to a complex group of proteins in bodyfluids that, working together with antibodies or other factors, play animportant role as mediators of immune allergic, immunochemical and/orimmunopathological reactions. The reactions in which complementparticipates takes place in blood serum or in other body fluids, andhence are considered to be humoral reactions.

With regard to human blood, there are at present more than 11 proteinsin the complement system. There complement proteins are designated bythe letter C and by number: C1, C2, C3 and so on up to C9. Thecomplement protein C1 is actually an assembly of subunits designatedC1q, C1r and C1s. The numbers assigned to the complement proteinsreflect the sequence in which they become active, with the exception ofcomplement protein C4, which reacts after C1 and before C2. Thenumerical assignments for the proteins in the complement system weremade before the reaction sequence was fully understood. A more detaileddiscussion of the complement system and its role in body processes canbe found in, for example, Bull. World Health Org., 39, 935-938 (1968);Scientific American, 229, (No. 5), 54-66 (1973); Medical World News,Oct. 11, 1974, pp. 53-58, 64-66; Harvey Lectures, 66, 75-104 (1972); TheNew England Journal of Medicine, 287, 489-495; 545-549; 592-596; 642-646(1972); The Johns Hopkins Med. J., 128, 57-74 (1971); and FederationProceedings, 32, 134-137 (1973).

The complement system can be considered to consist of three sub-systems:(1) a recognition unit (C1q) which enables it to combine with antibodymolecules that have detected a foreign invader; (2) an activation unit(C1r, C1s, C2, C4, C3), which prepares a site on the neighboringmembrane; and (3) an attack unit (C5, C6, C7, C8, and C9) which createsa "hole" in the membrane. The membrane attack unit is non-specific; itdestroys invaders only because it is generated in their neighborhood. Inorder to minimize damage to the host's own cells, its activity must belimited in time. This limitation is accomplished partly by thespontaneous decay of activated complement and partly by interference byinhibitors and destructive enzymes. The control of complement, however,is not perfect, and there are times when damage is done to the host'scells. Immunity is therefore a double-edged sword.

Activation of the complement system also accelerates blood clotting.This action comes about by way of the complement-mediated release of aclotting factor from platelets. The biologically active complementfragments and complexes can become involved in reactions that damage thehost's cells, and these pathogenic reactions can result in thedevelopment of immune-complex diseases. For example, in some forms ofnephritis complement damages the basal membrane of the kidney, resultingin the escape of protein from the blood into the urine. The diseasedisseminated lupus erythematosus belongs in this category; its symptomsinclude nephritis, visceral lesion and skin eruptions. The treatment ofdiphtheria or tetanus with the injection of large amounts of antitoxinsometimes results in serum sickness, an immune-complex diseases.Rheumatoid arthritis also involves immune complexes. Like disseminatedlupus erythematosus, it is an autoimmune disease, in which the diseasesymptoms are caused by pathological effects of the immune system in thehost's tissues. In summary, the complement system has been shown to beinvolved with inflammation, coagulation, fibrinolysis, antibody-antigenreactions and other metabolic processes.

In the presence of antibody-antigen complexes the complement proteinsare involved in a series of reactions which may lead to irreversiblemembrane damage if they occur in the vicinity of biological membranes.Thus, while complement constitutes a part of the body's defensemechanism against infection, it also results in inflammation and tissuedamage in the immunopathological process. The nature of certain of thecomplement proteins, suggestions regarding the mode of complementbinding to biological membranes and the manner in which complementeffects membrane damage are discussed in Annual Review in Biochemistry,38, 389 (1969).

A variety of substances have been disclosed as inhibiting the complementsystem, i.e., as complement inhibitors. For example, the compounds3,3'-ureylenebis[6-(2-amino-8-hydroxy-6-sulfo-1-naphthylazo)benzenesulfonicacid], tetrasodium salt (chlorazol fast pink), heparin and a sulphateddextran have been reported to have an anticomplementary effect, BritishJournal of Experimental Pathology, 33, 327-339 (1952). The compound8,8'-[ureylene[m-phenylenecarbonylimino(4-methyl-m-phenylene)carbonylimino]]di-1,3,5-naphthalenetrisulfonicacid, hexasodium salt (Suramin Sodium) as a competitive inhibitor of thecomplement system, Clin. Exp. Immunol., 10, 127-138 (1972). GermanPatent No. 2,254,893 or South African Pat. No. 727,923 discloses certain1-(diphenylmethyl)-4-(3-phenylallyl)piperazines useful as complementinhibitors. U.S. Pat. No. 3,897,434 discloses certainpyrazolo[1,5c]-quinazolin-5(6H)-ones useful as complement inhibitors.The compound m-[m-(p-nitrophenylureido)phenozypropoxyl]benzamidine isalso known as a complement inhibitor, Immunology, 26, 819 (1974). Otherchemical compounds having complement inhibiting activity are disclosedin, for example, Journal of Medicinal Chemistry, 12, 415-419; 902-905;1049-1052; 1053-1056 (1969); Canadian Journal of Biochemistry, 47,547-552 (1969); The Journal of Immunology, 93, 629-640 (1964); TheJournal of Immunology, 104, 279-288 (1970); The Journal of Immunology,106, 241-245 (1971); and The Journal of Immunology, 111, 1061-1066(1973).

It has been reported that the known complement inhibitorsepsilon-aminocaproic acid, Suramin Sodium and tranexamic acid have beenused with success in the treatment of hereditary angioneurotic edema, adisease state resulting from an inherited deficiency or lack of functionof the serum inhibitor of the activated first component of complement(C1 inhibitor), The New England Journal of Medicine, 286, 808-812(1972); Allergol, Et. Immunopath, II, 163-168 (1974); and J. AllergyClin. Immunol., 53, No. 5, 298-302 (1974).

SUMMARY OF THE INVENTION

Broadly, this invention is concerned with the use ofsubstituted-hydroxy-naphthalenedisulfonic acids, acid ureides and saltsthereof which can be represented by general formula (I): ##STR1## R₂ ishydrogen and --CH₃ ; R₃ is hydrogen, --NO₂, --NH₂ and --CH₃ ;

R₄ is hydrogen, --NO₂, --NH₂, --CH₃ and ##STR2## R₅ and R₆ are hydrogenand --CH₃ ; and A is hydrogen, alkali metal or alkaline earth, with theproviso that each A is identical in the same compound. Preferably, A issodium or potassium.

Of particular interest are those compounds encompassed within generalformula (I) which can be more specifically represented by generalformula (II): ##STR3## wherein R is selected from the group comprising##STR4## and R₅ and R₆ are hydrogen and --CH₃.

It has now been discovered that certain (substituted);4-hydroxy-2,7-naphthalenedisulfonic acids and4-hydroxy-2,7-naphthalenedisulfonic acid ureides interact with thecomplement reaction sequence, thereby inhibiting complement activity inbody fluids.

This invention is particularly concerned with new compounds and theiruse which can be represented by formula ##STR5## as well as with the useof known compounds which may be represented by formula (IV): ##STR6##wherein R₈ = H and CH₃ ; R₉ = H; NO₂ and CH₃ ; R₁₀ = H; NO₂ ; NH₂ ; CH₃and ##STR7## This invention is also concerned with the use of compoundsof formula (V): ##STR8## wherein R₁₁ = H and CH₃ ;

R₁₂ = and CH₃ ; and

M = so₃ na; SO₃ H

and with the use of the known compound of formula (VI): ##STR9##

The following references disclose representative compounds of thisinvention: J. Chem. Soc., 3068 (1927); J. Chem. Soc., 3739 (1956);Biochem. J., 42, 109 (1948); Biochem. J., 47, 158 (1950); and Ann. Inst.Pasteur, 38, 81 (1924).

This invention is specifically concerned with the following newcompounds and their use:4-hydroxy-5-{m-[3-(α,α,α-trifluoro-m-tolyl)ureido]benzamido}-2,7-naphthalenedisulfonicacid, disodium salt; and4-{m-{3-[p-(Fluorosulfonyl)phenyl]ureido}benzamido}-5-hydroxy-2,7-naphthalenedisulfonicacid, disodium salt. It is also concerned with the use of the followingknown compounds:4-hydroxy-5-(p-nitrobenzamido)-2,7-naphthalenedisulfonic acid, disodiumsalt; 4-(p-Aminobenzamido)-5-hydroxy-2,7-naphthalenedisulfonic acid;disodium salt;4-(4-Amino-m-toluamido)-5-hydroxy-2,7-naphthaleledisulfonic acid,disodium salt;4-(4-Amino-m-toluamido)-5-hydroxy-2,7-naphthalenedisulonic acid,disodium salt; 4-hydroxy-5-(m-nitrobenzamido)-2,7-naphthalenedisulfonicacid, disodium salt;4-(m-Aminobenzamido)-5-hydroxy-2,7-naphthalenedisulfonic acid, disodiumsalt;4-hydroxy-5-{m-[3-(3-nitro-p-tolyl)ureido]benzamido}-2,7-naphthalenedisulfonicacid, disodium salt;4-hydroxy-5-(5-nitro-o-toluamido)-2,7-naphthalenedisulfonic acid,disodium salt;4-(5-Amino-o-toluamido)-5-hydroxy-2,7-naphthalenedisulfonic acid,disodium salt;4-hydroxy-5-(3-nitro-p-toluamido)-2,7-naphthalenedisulfonic acid,disodium salt;5,5'-[Ureylenebis(m-phenylenecarbonylimino)]bis-[4-hydroxy-2,7-naphthalenedisulfonicacid], tetrasodium salt; 4,4'-{Ureylenebis[(2-methyl-1,3-phenylene)carbonyl]imino}bis[5-hydroxy-2,7-naphthalenedisulfonicacid]; and3,3'-[Ureylenebis(m-phenylenecarbonylimino)]bis[5-hydroxy-2,7-naphthalenedisulfonicacid], tetrasodium salt.

The compounds of this invention may be prepared as shown by the examplesherein and according to the methods described below. The compounds ofthe present invention may be prepared by acylation of the respectivenaphthylaminedisulfonic acid to the respective nitrobenzoyl derivativewith a nitrobenzoyl chloride. The aminobenzoyl derivative is then formedby catalytic reduction of the nitro group, condensation with phosgeneaffords the respective s-carbamide; or condensation with the appropriatesubstituted phenylisocyanate affords unsymmetrical areas. Acidificationproduces the free acid. The following will illustrate the preparation ofthe compounds in more detail.

Schotten-Baumann acylation

To a solution of the sodium salt of a naphthylaminedisulfonic acid in anappropriate amount of water and 1N sodium hydroxide is added anappropriate nitrobenzoyl chloride. The mixture is shaken until no longerbasic to test paper. Three additional equal portions of 1N sodiumhydroxide are added, shaking between each addition until the solution isno longer basic. After the last portion of base is added, the reactionmixture is shaken for at least 30 minutes and then the still basicsolution is acidified to Congo Red with concentrated hydrochloric acid.The reaction mixture is then copiously extracted with ether to removethe nitrobenzoic acid side product (by vacuum siphoning of the ethereallayer). The aqueous phase is then filtered to remove a small amount ofthe anhydride of the particular nitrobenzoic acid and the filtrate isconcentrated in vacuo at 50°-60° C. until a solid is precipitated. Aftercooling to ambient temperature, the product is filtered and is washedwith saturated saline solution, 50% ethyl alcohol, absolute ethylalcohol and ether.

Catalytic reduction

Treatment of a solution of the appropriate amount of the desiredm-nitrobenzamide of naphthalenedisulfonic acid in 160-200 ml of waterwith 1.0-3.7 g of 10% palladium on carbon in a Parr apparatus under aninitial hydrogen pressure of 42 pounds per square inch gives atheoretical uptake of hydrogen in 13/4 hours. The reaction mixture isfiltered through diatomaceous earth and the catalyst is washed withwater. The filtrate is concentrated under reduced pressure at 50°-60° C.to low volume and is then diluted with a large volume of absoluteethanol. The precipitated product is collected and is washed withabsolute ethyl alcohol.

Phosgenation

Phosgene is bubbled through a mechanically stirred solution of thedesired aminobenzamide of naphthalenedisulfonic acid in the appropriateamount of water containing a theoretical quantity of sodium carbonateuntil the reaction mixture becomes acidic to Congo Red. An additionalquantity of carbonate is cautiously added and the process is repeateduntil the reaction mixture is again acidic. It is then neutralized withbicarbonate and is concentrated in vacuo at 50°- 60° C. On cooling toroom temperature a solid is formed which is filtered and is washed with80% ethyl alcohol, absolute ethyl alcohol and ether.

Acylation with isocyanates

A solution of the appropriate amount of the desired m-aminobenzamide ofnaphthalenedisulfonic acid sodium salt in water is treated with atheoretical portion of the required isocyanate and is stirred vigorouslyfor 6 hours at room temperature. The reaction mixture is diluted withadditional water, is heated to approximatley 95° C. to 30 minutes and isfiltered through diatomaceous earth and is washed with hot water. Thefiltrate is treated with sodium chloride while heating on the steam bathand is allowed to stand at room temperature overnight (lower temperatureis required in some cases). The precipitate is collected and is boiledwith absolute ethyl alcohol then is allowed to stand at room temperaturefor several days. The product is then filtered and washed with absoluteethyl alcohol and ether.

This invention is concerned with a method of inhibiting the complementsystem in a body fluid, such as blood serum, which comprises subjectingbody fluid complement to the action of an effective complementinhibiting amount of a compound encompassed within the formulaehereinabove. The method of use aspect of this invention is alsoconcerned with a method of inhibiting the complement system in awarm-blooded animal which comprises internally administering to saidanimal an effective complement inhibiting amount of a compoundencompassed within the formulae hereinabove. Body fluid can includeblood, plasma, serum, synovial fluid, cerebrospinal fluid, orpathological accumulations of fluid as pleural effusion, etc.

Compounds of the present invention find utility as complement inhibitorsin body fluids and as such may be used to ameliorate or prevent thosepathological reactions requiring the function of complement and in thetherapeutic treatment of warm-blooded animals having immunologicdiseases such as rheumatoid arthritis, systemic lupus erythematosus,certain kinds of glomerulonephritis, certain kinds of auto-allergichemolytic anemia, certain kinds of platelet disorders and certain kindsof vasculitis. The compounds herein may also be used in the therapeutictreatment of warm-blooded animals having non-immunologic diseases suchas paroxysmal nocturnal hemoglobinuria, hereditary angioneurotic edema(treated with Suramin, etc.) and inflammatory states induced by theaction of bacterial or lysosomal enzymes on the appropriate complementcomponents as for example, inflammation following coronary occlusion.They may also be useful in the treatment of transplant rejection and asblood culture or transport mediums.

DETAILED DESCRIPTION OF THE INVENTION

The following examples will serve to illustrate the invention in moredetail.

EXAMPLE 1 4-Hydroxy-5-(4-nitro-3-toluamido)-2,7-naphthalenedisulfonicacid, disodium salt

A 76.5 g portion of 3-methyl-4-nitrobenzoic acid in 160 ml of thionylchloride is refluxed for 23/4 hours (with complete solution occuringafter 35 minutes). The solution is evaporated to an oil in vacuo and isreevaporated several times from toluene. The material is vacuumdistilled at a bath temperature of 140°-150° C. and at about 0.6 mmpressure. After rejecting the first few ml the fraction boiling at107°-110° C. is collected to give 81.1 g of 4-nitro-m-toluoyl chloride.

A 26.9 g portion of recrystallized 8-amino-1-naphthol-3,6-disulfonicacid monosodium salt is suspended in 125 ml of water along with 10.0 gof sodium acetate trihydrate, then 75 ml of 1N sodium hydroxide is addedand 16.5 g of the product prepared above is added all at once, washingin with a small amount of diethyl ether. The mixture is shaken for 10minutes and the addition of 1N sodium hydroxide with shaking is repeatedtwice, the final shake is continued for 45 minutes. The mixture is thenacidified with 11 ml of concentrated hydrochloric acid and is extractedwith six 150 ml portions of ether, the ether is removed each time byvacuum siphoning. The aqueous phase is neutralized with base and 6 dropsof n-decyl alcohol is added to prevent frothing. The solution isconcentrated in vacuo at about 55°-60° C. until a solid begins toprecipitate. The mixture is allowed to stand overnight at roomtemperature and is filtered. The precipitate collected is washed with80% ethyl alcohol followed by absolute ethyl alcohol and ether. Theproduct is dried overnight at 120° C. then is dissolved in about 200 mlof boiling water and is diluted with 100 ml of absolute ethyl alcoholfollowed by 200 ml of 331/3% ethyl alcohol. The thick mixture istriturated and allowed to stand overnight then is filtered and theproduct of the example is washed with 80% ethyl alcohol followed byabsolute ethyl alcohol and ether.

EXAMPLE 2 4-(4-Amino-m-toluamido)-5-hydroxy-2,7-naphthalenedisulfonicacid, disodium salt

A mixture of 25.0 g of4-hydroxy-5-(4-nitro-3-toluamido)-2,7-naphthalenedisulfonic acid,disodium salt, 200 ml of distilled water and 2.5 g of 10% palladium oncharcoal is hydrogenated in a Parr shaker for 3 hours at roomtemperature during which time 12 pounds of hydrogen is absorbed. Themixture is heated to dissolve some precipitated product and is filteredhot to remove the catalyst. The filtrate is then evaporated to a lowvolume in vacuo at 55°-60° C. and is diluted with a large amount ofabsolute ethyl alcohol. The precipitate formed is triturated with aglass rod to break up any lumps and is allowed to stand at roomtemperature overnight then is filtered and is washed with absolute ethylalcohol and ether. The product of the example is then oven dried at 120°C. overnight.

EXAMPLE 3 4-Hydroxy-5-(m-nitrobenzamido)-2,7-naphthalenedisulfonic acid,disodium salt

A 44.1 g portion of recrystallized4-amino-5-hydroxy-2,7-naphthalenedisulfonic acid, mono sodium salt and16.5 g of sodium acetate trihydrate is suspended in 40 ml of distilledwater, then 120 ml of 1N sodium hydroxide is added and 44.55 g ofm-nitrobenzoyl chloride is added all at once washing in with a smallamount of diethyl ether. The resulting mixture is shaken for 5 minutesand another 120 ml of 1N sodium hydroxide is added and the mixture isshaken for 1/2 hour. The latter addition and shaking is repeated twomore times. The resulting clear solution is acidified to Congo Redindicator paper with concentrated hydrochloric acid and is concentratedin vacuo at about 55° C. until a precipitate forms which is collected byfiltration. The filtrate is set aside and the precipitate is ground upand is stirred with about 400 ml of ether for 20 minutes then isfiltered and the precipitate is set aside. The filtrate previously setaside is extracted 3 times with ether which is removed by vacuumsiphoning after each extraction. The aqueous solution is then kept at 5°C. overnight with formation of a precipitate which is also collected.The precipitate set aside above is combined with the present precipitateand is dissolved in 400 ml of boiling water. A 100 ml portion of 95%ethyl alcohol is added and the solution is kept at 5° C. overnight withprecipitation of the product of the example which is collected byfiltration.

EXAMPLE 4 4-hydroxy-5-(p-nitrobenzamido-2,7-naphthalenedisulfonic aciddisodium salt

In a manner similar to that of Example 3,4-hydroxy-5-(p-nitrobenzamido-2,7-naphthalenedisulfonic acid disodiumsalt can be prepared from p-nitrobenzoyl chloride.

EXAMPLE 5 4-(m-Aminobenzamido)-5-hydroxy-2,7-naphthalenedisulfonic acid,disodium salt

A mixture of 15.89 g of4-hydroxy-5-(m-nitrobenzmido)-2,7-naphthalenedisulfonic acid, disodiumsalt, 150 ml of distilled water and 1.0 g of 10% palladium on charcoalis hydrogenated in a Parr shaker for 2 hours at room temperature duringwhich time 7.5 pounds of hydrogen is absorbed. The mixture is heated todissolve some precipitated product and is filtered hot to remove thecatalyst. The filtrate is then evaporated to a low volume in vacuo at55°-60° C. and is diluted with a large amount of absolute ethyl alcohol.The precipitate formed is triturated with a glass rod to break up anylumps and is allowed to stand at room temperature overnight, then isfiltered and is washed with absolute ethyl alcohol and ether to give theproduct of the example.

EXAMPLE 6 4-(p-aminobenzamido)-5-hydroxy-2,7-naphthalenedisulfonic aciddisodium salt

In a manner similar to that of Examples 3 and 54-(p-aminobenzamido)-5-hydroxy-2,7-naphthalenedisulfonic acid disodiumsalt can be prepared.

EXAMPLE 74-Hydroxy-5-m-[3-(α,α,α-trifluoro-m-tolyl)ureido]benzamido-2,7-naphthalenedisulfonicacid, disodium salt

A mixture of 3.0 g of4-(m-amino-benzamido)-5-hydroxy-2,7-naphthalenedisulfonic acid, disodiumsalt, 37.5 ml of distilled water and 3.0 ml ofm-trifluoromethylphenylisocyanate is stirred for 6 hours, then is heatedon a steam bath and is filtered through diatomaceous earth and thefilter is washed with hot water. The volume of filtrate is adjusted to60 ml and is salted with 10 g of sodium acetate then is allowed to standat room temperature overnight. The precipitate formed is collected byfiltration and is washed on the filter with a small amount of absoluteethyl alcohol and a large amount of diethyl ether. The material is thenoven dried at 120° C. for 2 hours and is then redissolved in 35-40 ml ofhot water. This solution is kept at 5° C. overnight. The resultingprecipitate is collected by filtration and is dried as above to give theproduct of the example.

EXAMPLE 84-{m-{3-[p-(Fluorosulfonyl)phenyl]ureido}benzamido}-5-hydroxy-2,7-naphthalenedisulfonicacid, disodium salt

A mixture of 18.0 g of sulfanilyl fluoride, 20.0 g of p-nitrophenylchloroformate and 95 ml of benzene (dried by distillation) is refluxedand stirred for 5 hours (a precipitate appears at about 4 hours). Themixture is then cooled and filtered, the precipitate is washed with coldbenzene and is recrystallized from methylene chloride to give 19.86 g ofp-(fluorosulfonyl)carbanilic acid p-nitrophenyl ester.

A mixture of 4.0 g of4-(m-amino-benzamido)-5-hydroxy-2,7-naphthalenedisulfonic acid, disodiumsalt in 40 ml of dimethylformamide (dried over 4A molecular sieve) and16 g of 3A molecular sieve (Linde-type 1/16 inch) is stirred one hour atroom temperature, then 3.12 g of p-(fluorosulfonyl)carbanilic acidp-nitrophenyl ester in 16 ml of dried dimethylformamide is added andstirring is continued for an additional 18 hours at room temperature.The resulting mixture is then filtered through diatomaceous earth andthe filter is washed with dried dimethylformamide. The combined filtrateand washing is poured into about one liter of diethyl ether resulting information of a solid which is collected by filtration to give theproduct of the example.

EXAMPLE 94-Hydroxy-5-m-[3-(3-nitro-p-tolyl)ureido]benzamido-2,7-naphthalenedisulfonicacid, disodium salt

A mixture of 3.0 g of4-(m-amino-benzamido)-5-hydroxy-2,7-naphthalenedisulfonic acid, disodiumsalt in 37.5 ml of distilled water and 3.0 g of 3-nitro-4-methylphenylisocyanate is stirred for 6 hours to give a gelatenous mass which iscentrifuged. The clear liquid is decanted off and the residue isfiltered using absolute ethyl alcohol to wash the material in thefilter. The product of the example is oven dried at 120° C.

EXAMPLE 10 4Hydroxy-5-(5-nitro-o-toluamido)-2,7-naphthlenedisulfonicacid, disodium salt

A mixture of 74.5 g of 2-methyl-5-nitrobenzoic acid and 160 ml ofthionyl chloride is refluxed for 41/2 hours (complete solution after11/2 hours). The mixture is evaporated in vacuo to an oil then isreevaporated several times with toluene, finally about 300 ml of hexaneis added resulting in formation of needles after standing at roomtemperature overnight. The material is collected by filtration and iswashed with hexane to give 5-nitro-o-toluoyl chloride.

A 26.9 g portion of recrystallized4-amino-5-hydroxy-2,7-naphthalenedisulfonic acid, mono sodium salt and10.0 g of sodium acetate trihydrate is suspended in 125 ml of distilledwater, then 75 ml of 1N sodium hydroxide is added and 16.5 g of5-nitro-o-toluoyl chloride (prepared above) is added all at once washingin with a small amount of diethyl ether. The resulting mixture is shakenfor 5 minutes. The addition of two more 75 ml portions of 1N sodiumhydroxide is required with shaking for 45 minutes after the lastaddition. The mixture is then acidified with 13 ml of concentratedhydrochloric acid and is extracted with six 150 ml portions of diethylether which is removed by vacuum siphoning after each extraction. Theaqueous phase is then neutralized with base and 6 drops of n-decylalcohol is added to prevent frothing. The solution is then concentratedto a low volume in vacuo and is diluted with a saturated saline solutionto produce a precipitate. The precipitated product is filtered veryslowly and is washed with absolute ethyl alcohol followed by ether. Thelumpy material is oven dried at 120° C. for several hours then isdissolved in about 600 ml of boiling water and is diluted with 150 ml ofabsolute alcohol. The precipitate formed after standing at roomtemperature overnight is collected by filtration and is washed withabsolute ethyl alcohol followed by ether then is oven dried overnight at120° C. to give the product of the example.

EXAMPLE 11 4-(5-Amino-o-toluamido)-5-hydroxy-2,7-naphthalenedisulfonicacid, disodium salt

A mixture of 25.0 g of4-hydroxy-5-(5-nitro-o-toluamido)-2,7-naphthalenedisulfonic acid,disodium salt, 200 ml of distilled water and 2.5 g of 10% palladium oncharcoal is hydrogenated in a Parr shaker for 5 hours at roomtemperature during which time 12 pounds of hydrogen is absorbed. Themixture is heated on a steam bath and is filtered through diatomaceousearth to remove the catalyst. The filter is washed with hot water andthe filtrate is then evaporated to about 100 ml in vacuo at 55°-60° C.with formation of crystals after standing at room temperature overnight.The product of the example is collected by filtration and is washed withabsolute ethyl alcohol followed by ether then is oven dried at 120° C.

EXAMPLE 12 4-Hydroxy-5-(3-nitro-p-toluamido)-2,7-naphthalenedisulfonicacid, disodium salt

A mixture of 75.3 of 4-methyl-3-nitrobenzoic acid and 150 ml of thionylchloride is refluxed for 41/2 hours in a 120° C. oil bath (completesolution is achieved after about 21/4 hours). The mixture is evaporatedin vacuo to an oil then is reevaporated several times with toluene. Thematerial is then distilled at a bath temperature of 150° C. and about0.6 mm. The fraction collected at 105° -110° C. is4-methyl-3-nitrobenzoylchloride.

A 26.9 g portion of recrystallized4-amino-5-hydroxy-2,7-naphthalenedisulfonic acid, mono sodium salt and10.0 g of sodium acetate trihydrate is suspended in 125 ml of distilledwater, then 75 ml of 1N sodium hydroxide is added and 16.5 g of4-methyl-3;l -nitrobenzoyl chloride is added all at once washing in witha small amount of diethyl ether. The procedure from this point on isidentical to that in Example 8 with the exception that 11 ml ofconcentrated hydrochloric acid is used to acidify the mixture.

EXAMPLE 13 4-(3-Amino-p-toluamido)-5-hydroxy-2,7-naphthalenedisulfonicacid, disodium salt

A mixture of 25.0 g of4-hydroxy-5-(3-nitro-p-toluamido)-2,7-naphthalenedisulfonic acid,disodium salt, 200 ml of distilled water and 2.5 g of 10% palladium oncharcoal is hydrogenated in a Parr shaker for 31/2 hours at roomtemperature during which time 12 pounds of hydrogen is absorbed. Themixture is heated on a steam bath and is filtered through diatomaceousearth to remove the catalyst. The filter is washed with hot water andthe filtrate is then evaporated to a low volume in vacuo at 55°-60° C.and diluted with a large amount of absolute ethyl alcohol. Theprecipitate formed is triturated with a glass rod to break up any lumpsand is allowed to stand at room temperature overnight, then is filteredand is washed with absolute ethyl alcohol and ether. The product of theexample is oven dried at 120° C. overnight.

EXAMPLE 145,5'-[Ureylenebis(m-phenylenecarbonylimino)]bis[4-hydroxy-2,7-naphthalenedisulfonicacid] tetrasodium salt

A mixture of 11.32 g of4-hydroxy-5-m-nitrobenzamido-2,7-naphthalenedisulfonic acid, disodiumsalt, 150 ml of distilled water and 0.5 g of 10% palladium on charcoalis hydrogenated in a Parr shaker for 3 hours at room temperature duringwhich time 13 pounds of hydrogen is absorbed. The mixture is heated on asteam bath and is filtered through diatomaceous earth to remove thecatalyst. The filter is washed with hot water and the filtrate is cooledto room temperature then 1.83 g of concentrated hydrochloric acid isadded and the precipitate formed is dissolved by boiling. The aqueous isconcentrated to about 175 ml and 100 ml of absolute ethyl alcohol isadded and the resultant mixture is allowed to come to room temperatureovernight. The precipitate formed which is4-m-amino-benzamido-5-hydroxy-2,7-naphthalenedisulfonic acid 2-sodiumsalt is collected by filtration.

To a stirred solution of 10 g of the above product prepared in the samemanner as described above and 34.5 g of anhydrous sodium carbonate in250 ml of water is bubbled in phosgene at ambient temperature. After 2hours an additional 17 g of sodium carbonate is added and the phosgeneaddition is continued for another hour. The precipitate formed iscollected then is mixed with water and is neutralized with base, then isheated and filtered. The material collected is dissolved in 300 ml ofhot water then is concentrated to 200 ml in vacuo. The gelatinousprecipitate is collected by slow filtration then is stirred with 95%ethyl alcohol. The resulting product is filtered and is washed withdiethyl ether to give the product of the example.

EXAMPLE 153,3'-{Ureylenebis[(4-methyl-1,3-phenylene)carbonyl]}bis[2,3-dihydro-2-oxo-naphth[1,8-de]-1,3-oxazine-5,8-disulfonicacid] tetrasodium salt

To a stirred solution of 10.0 g of4-(5-amino-o-toluamido)-5-hydroxy-2,7-naphthalenedisulfonic acid,disodium salt (prepared as described in Example 9) and 21.4 g ofanhydrous sodium carbonate in 250 ml of water is bubbled in phosgene forone hour at room temperature. Then an additional 21.4 g of sodiumcarbonate is added and phosgenation is continued for one hour and 25minutes. The resulting mixture is neutralized with 16.5 g of sodiumcarbonate and is filtered and the filtrate is set aside for use inExample 14. The precipitate is washed with a small amount of water thenis oven dried at 120° C. for several hours to give the product of theexample.

EXAMPLE 164,4'-{Ureylenebis[(2-methyl-1,3-phenylene)carbonyl]imino}bis[5-hydroxy-2,7-naphthalenedisulfonicacid]

The aqueous phase filtrate set aside at the conclusion of Example 13 isconcentrated in vacuo at about 50°-60° C. and is filtered. The filtrateis evaporated to dryness and is triturated with about 250 ml of boilingmethyl alcohol then is filtered. This filtrate is then evaporated invacuo to afford a brown solid which is dissolved in 10 ml of distilledwater, the solution is then warmed on a steam bath and is diluted withabout 30 ml of absolute ethyl alcohol to give a black viscous material.The supernatent is decanted and absolute alcohol is added to the residuewith scratching to afford a precipitate which is collected byfiltration. This material is dissolved in 10 ml of distilled water, isboiled then is treated with activated charcoal and filtered throughdiatomaceous earth. The filtrate is diluted 2 fold with absolute ethylalcohol and the upper phase is decanted from a dark oil. The decantedliquid is acidified with concentrated hydrochloric acid and isevaporated to dryness in vacuo. The residue is dissolved in 5 ml ofdimethylformamide and is filtered to remove sodium chloride, then iswashed with 5 ml of dimethylformamide. The combined filtrate and washingis diluted with a few ml of methyl alcohol and a large volume of diethylether to yield a dark brown oil. The supernatent is decanted and acetoneis added to the residue with scratching until a solid forms which iscollected by filtration and is washed with acetone. The product of theexample is then oven dried at 105° C. overnight.

EXAMPLE 173,3'-[Ureylenebis(m-phenylenecarbonylimino)]bis[5-hydroxy-2,7-naphthalenedisulfonicacid] tetrasodium salt

A 41.0 g portion of recrystallized3-amino-5-hydroxy-2,7-naphthalenedisulfonic acid and 16.5 g of sodiumacetate trihydrate is dissolved in 250 ml of distilled water, then 44.55g of m-nitrobenzoyl chloride is added all at once along with 120 ml of1N sodium hydroxide and a small amount of diethyl ether. The resultingmixture is shaken for 5 minutes and another 120 ml of 1N sodiumhydroxide is added and the mixture is shaken for 1/2 hour. The latteraddition and shaking is repeated 2 more times. The resulting mixture isacidified to Congo Red indicator paper with concentrated hydrochloricacid and is allowed to stand at 5° C. for 17 hours. The solution isfiltered and the filtrate is allowed to stand until a precipitate forms.The precipitate is stirred with about 400 ml of diethyl ether and iscollected to give 5-hydroxy-3-m-nitrobenzamido-2,7-naphthalenedisulfonicacid, disodium salt.

A mixture of 15.5 g of the preceding compound (prepared in a manner asdescribed above), 170 ml of distilled water and 1.02 g of 10% palladiumon charcoal is hydrogenated in a Parr shaker for 11/2 hours at roomtemperature during which time 8.5 pounds of hydrogen is absorbed. Themixture is heated to dissolve some precipitated product and is filteredhot to remove the catalyst. The filtrate is then evaporated to a lowvolume and the precipitate formed is collected with the aid of 95% ethylalcohol and is washed with diethyl ether to give3-(m-aminobenzamido)-5-hydroxy-2,7-naphthalenedisulfonic acid, disodiumsalt. To a stirred solution of 10 g of the above product prepared in themanner described above and 34.5 g of anhydrous sodium carbonate in 250ml of water is bubbled in phosgene at ambient temperature. After onehour and 15 minutes an additional 34.5 g of sodium carbonate is addedand the phosgene addition is continued until it is acidic again (5hours). The solution is then neutralized with base and is filtered. Thefiltrate is concentrated and filtered and the final filtrate isconcentrated and filtered. All the residues are combined and dissolvedin 250 ml of hot water which is concentrated to 150 ml, is cooled andthe precipitate formed is collected by filtration. The precipitate isdissolved in hot water and is concentrated to a low volume until aprecipitate is formed which is collected by filtration with the aid of95% ethyl alcohol to give the product of the example.

                  Example 18                                                      ______________________________________                                        Preparation of Compressed Tablet                                              Ingredient            mg/Tablet                                               ______________________________________                                        Active Compound       0.5-500                                                 Dibasic Calcium Phosphate NF                                                                        qs                                                      Starch USP            40                                                      Modified Starch       10                                                      Magnesium Stearate USP                                                                              1-5                                                     ______________________________________                                    

                  Example 19                                                      ______________________________________                                        Preparation of Compressed Tablet-Sustained Action                             Ingredient           mg/Tablet                                                ______________________________________                                        Active Compound      0.5-500(as acid                                          as Aluminum Lake*, Micronized                                                                      equivalent)                                              Dibasic Calcium Phosphate NF                                                                       qs                                                       Alginic Acid         20                                                       Starch USP           35                                                       Magnesium Stearate USP                                                                              1-10                                                    ______________________________________                                         *Complement inhibitor plus aluminum sulfate yields aluminum complement        inhibitor. Complement inhibitor content in aluminum lake ranges from          5-30%.                                                                   

                  Example 20                                                      ______________________________________                                        Preparation of Hard Shell Capsule                                             Ingredient          mg/Capsule                                                ______________________________________                                        Active Compound      0.5-500                                                  Lactose, Spray Dried                                                                              qs                                                        Magnesium Stearate   1-10                                                     ______________________________________                                    

                  Example 21                                                      ______________________________________                                        Preparation of Oral Liquid (Syrup)                                            Ingredient          % W/V                                                     ______________________________________                                        Active Compound     0.05-5                                                    Liquid Sugar        75.0                                                      Methyl Paraben USP  0.18                                                      Propyl Paraben USP  0.02                                                      Flavoring Agent     qs                                                        Purified Water qs ad                                                                              100.0                                                     ______________________________________                                    

                  Example 22                                                      ______________________________________                                        Preparation of Oral Liquid (Elixir)                                           Ingredient          % W/V                                                     ______________________________________                                        Active Compound     0.05-5                                                    Alcohol USP         12.5                                                      Glycerin USP        45.0                                                      Syrup USP           20.0                                                      Flavoring Agent     qs                                                        Purified Water qs ad                                                                              100.0                                                     ______________________________________                                    

                  Example 23                                                      ______________________________________                                        Preparation of Oral Suspension (Syrup)                                        Ingredient           % W/V                                                    ______________________________________                                        Active Compound      0.05-5                                                   as Aluminum Lake, Micronized                                                                       (acid equivalent)                                        Polysorbate 80 USP   0.1                                                      Magnesium Aluminum Silicate,                                                  Colloidal            0.3                                                      Flavoring Agent      qs                                                       Methyl Paraben USP   0.18                                                     Propyl Paraben USP   0.02                                                     Liquid Sugar         75.0                                                     Purified Water qs ad 100.0                                                    ______________________________________                                    

                  Example 24                                                      ______________________________________                                        Preparation of Injectable Solution                                            Ingredient             % W/V                                                  ______________________________________                                        Active Compound        0.05-5                                                 Benzyl Alcohol NF      0.9                                                    Water for Injection    100.0                                                  ______________________________________                                    

                  Example 25                                                      ______________________________________                                        Preparation of Injectable Oil                                                 Ingredient         %                                                          ______________________________________                                        Active Compound    0.05-5                                                     Benzyl Alcohol     1.5                                                        Sesame Oil qs ad   100.0                                                      ______________________________________                                    

                  Example 26                                                      ______________________________________                                        Preparation of Intra-Articular Product                                        Ingredient             Amount                                                 ______________________________________                                        Active Compound        2-20 mg                                                NaCl (physiological saline)                                                                          0.9%                                                   Benzyl Alcohol         0.9%                                                   Sodium Carboxymethylcellulose                                                                        1-5%                                                   pH adjusted to 5.0-7.5                                                        Water for Injection qs to                                                                            100%                                                   ______________________________________                                    

                  Example 27                                                      ______________________________________                                        Preparation of Injectable Depo suspension                                     Ingredient           % W/V                                                    ______________________________________                                        Active Compound      0.05-5                                                                        (acid equivalent)                                        Polysorbate 80 USP   0.2                                                      Polyethylene Glycol 4000 USP                                                                       3.0                                                      Sodium Chloride USP  0.8                                                      Benzyl Alcohol NF    0.9                                                      HCl to pH 6-8        qs                                                       Water for Injection qs ad                                                                          100.0                                                    ______________________________________                                    

The compounds of this invention may be administered internally, e.g.,orally, or patenterally, e.g., intra-articularly, to a warm-bloodedanimal to inhibit complement in the body fluid of the animal, suchinhibition being useful in the amelioration or prevention of thosereactions dependent upon the function of complement, such asinflammatory process and cell membrane damage induced byantigen-antibody complexes. A range of doses may be employed dependingon the mode of administration, the condition being treated and theparticular compound being used. For example, for intravenous orsubcutaneous use from about 5 to about 50 mg/kg/day, or every six hoursfor more rapidly excreted salts, may be used. For intra-articular usefor large joints such as the knee, from about 2 to about 20 mg/joint perweek may be used, with proportionally smaller doses for smaller joints.The dosage range is to be adjusted to provide optimum therapeuticresponse in the warm-blooded animal being treated. In general, theamount of compound administered can vary over a wide range to providefrom about 5 mg/kg to about 100 mg/kg of body weight of animal per day.The usual daily dosage for a 70 kg subject may vary from about 350 mg toabout 3.5 g. Unit doses of the acid or salt can contain from about 0.5mg to about 500 mg.

In therapeutic use the compounds of this invention may be administeredin the form of conventional pharmaceutical compositions. Suchcompositions may be formulated so as to be suitable for oral orparenteral administration. The active ingredient may be combined inadmixture with a pharmaceutically acceptable carrier, which carrier maytake a wide variety of forms depending on the form of preparationdesired for administration, i.e., oral or parenteral. The compounds canbe used in compositions such as tablets. Here, the principal activeingredient is mixed with conventional tabletting ingredients such ascorn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesiumstearate, dicalcium phosphate, gums, or similar materials as non-toxicpharmaceutically acceptable diluents or carriers. The tablets or pillsof the novel compositions can be laminated or otherwise compounded toprovide a dosage form affording the advantage of prolonged or delayedaction or predetermined successive action of the enclosed medication.For example, the tablet or pill can comprise an inner dosage and anouter dosage component, the latter being in the form of an envelope overthe former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permits theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids ormixtures of polymeric acids with such materials as shellac, shellac andcetyl alcohol, cellulose acetate and the like. A particularlyadvantageous enteric coating comprises a styrene maleic acid copolymertogether with known materials contributing to the enteric properties ofthe coating. The tablet or pill may be colored through the use of anappropriate non-toxic dye, so as to provide a pleasing appearance.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration include suitableflavored emulsions with edible oils, such as, cottonseed oil, sesameoil, coconut oil, peanut oil, and the like, as well as elixirs andsimilar pharmaceutical vehicles. Sterile suspensions or solutions can beprepared for parenteral use. Isotonic preparations containing suitablepreservatives are also desirable for injection use.

The term dosage form as described herein refers to physically discreteunits suitable as unitary dosage for warm-blooded animal subjects, eachunit containing a predetermined quantity of active component calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical diluent carrier or vehicle. The specificationfor the novel dosage forms of this invention are indicated bycharacteristics of the active component and the particular therapeuticeffect to be achieved or the limitations inherent in the art ofcompounding such an active component for therapeutic use in warm-bloodedanimals as disclosed in this specification. Examples of suitable oraldosage forms in accord with this invention are tablets, capsules, pills,powder packets, granules, wafers, cachets, teaspoonfuls, dropperfuls,ampules, vials, segregated multiples of any of the foregoing and otherforms as herein described.

The complement inhibiting activity of representative compounds of thisinvention has been demonstrated by one or more of the followingidentified tests: (i) Test, Code 026 (C1 inhibitor). This test measuresthe ability of activated human fluid phase human C2 in the presence ofC4 and appropriate dilutions of the test compound. An active inhibitorprotects C2 from C1 and C4; (ii) Test, Code 035 (C3-C9 inhibitor) --This test determines the ability of the late components of humancomplement (C3-C9) to lyse EAC 142 in the presence of appropriatedilutions of the test compound. An active inhibitor protects EAC 142from lysis by human C3-C9; (iii) Test, Code 036 (C-Shunt inhibitor) --In this test human erythrocytes rendered fragile are lysed in autologousserum via the shunt pathway activated by cobra venom factor in thepresence of appropriate dilutions of the test compound. Inhibition ofthe shunt pathway results in failure of lysis; (iv) Forssman VasculitisTest -- Here, the well known complement dependent lesion, Forssmanvasculitis, is produced in guinea pigs by intradermal injection ofrabbit anti-Forssman antiserum. The lesion is measured in terms ofdiameter, edema and hemorrhage and the extent to which a combined indexof these is inhibited by prior intraperitoneal injection of the testcompound at 200 mg/kg is then reported, unless otherwise stated; (v)Forssman Shock Test -- Lethal shock is produced in guinea pigs by ani.v. injection of anti-Forssman antiserum and the harmonic mean deathtime of treated guinea pigs is compared with that of simultaneouscontrols; (vi) Complement Level Reduction Test -- In this test, theabove dosed quinea pigs, or others, are bled for serum and thecomplement level is determined in undiluted serum by the capillary tubemethod of U.S. Pat. No. 3,876,376 and compared to undosed control guineapigs; and (vii) Cap 50 Test -- Here, appropriate amounts of the testcompound are added to a pool of guinea pig serum in vitro, after whichthe undiluted serum capillary tube assay referred to above is run. Theconcentration of compound inhibiting 50% is reported.

Table I shows that representative compounds of the invention possesscomplement inhibitory activity.

                                      TABLE I                                     __________________________________________________________________________    Biological Activities                                                                                  Assay Results                                                                 In Vitro    In Vivo                                                                            % Reduction                         Compound                 026*                                                                              035 036 Forssman                                                                           Complement                          __________________________________________________________________________    4-Hydroxy-5-{m-[3-(α,α,α-trifluoro-m-tolyl)-                ureido]benzamido}-2,7-naphthalenedisulfonic                                                            1** NEG NEG 5    -6                                  acid, disodium salt                                                           4-{m-{3-[p-(Fluorosulfonyl)phenyl]ureido} -                                   benzamido}-5-hydroxy-2,7-naphthalenedisul-                                                             4   NEG NEG --   --                                  fonic acid, disodium salt                                                     4-Hydroxy-5-(4-nitro-3-toluamido)-2,7-naph-                                   thalenedisulfonic acid, disodium salt                                                                  2   NEG NEG 48   -20                                 4-(4-Amino-m-toluamido)-5-hydroxy-2,7-naph-                                   thalenedisulfonic acid, disodium salt                                                                  5   NEG NEG 17   +12                                 4-Hydroxy-5-{m-[3-(3-nitro-p-tolyl)ureido]-                                   benzamido}-2,7-naphthalenedisulfonic acid,                                                             NEG NEG NEG 15   +46                                 disodium salt                                                                 4-Hydroxy-5-(5-nitro-o-toluamido)-2,7-naph-                                   thalenedisulfonic acid, disodium salt                                                                  2   NEG NEG 10   -17                                 4-(5-Amino-o-toluamido)-5-hydroxy-2,7-naph-                                   thalenedisulfonic acid, disodium salt                                                                  2   NEG NEG --   --                                  4-Hydroxy-5-(3-nitro-p-toluamido)-2,7-naph-                                   thalenedisulfonic acid, disodium salt                                                                  3   NEG NEG --   --                                  4-(3-Amino-p-toluamido)-5-hydroxy-2,7-naph-                                   thalenedisulfonic acid, disodium salt                                                                  5   NEG NEG --   --                                  5,5'-[Ureylenebis(m-phenylenecarbonylimino)]-                                 bis[4-hydroxy-2,7-naphthalenedisulfonic acid]                                                          6   1   2   --   --                                  tetrasodium salt                                                              4,4'-{Ureylenebis[ (2-methyl-1,3-phenylene)-                                  carbonyl]imino}bis[5-hydroxy-2,7-naphthalene-                                                          7   3   3   --   --                                  disulfonic acid]                                                              3,3'-[Ureylenebis(m-phenylenecarbonylimino)]-                                 bis[5-hydroxy-2,7-naphthalenedisulfonic acid],                                                         5   2   2   35   -31                                 tetrasodium salt                                                              __________________________________________________________________________     *Tests identified by code herein.                                             **Numbers represent activity in wells. a serial dilution assay, higher        well number indicates higher activity. The serial dilutions are two-fold.

We claim:
 1. A compound selected from those of the formula: ##STR10##and A is hydrogen, alkali metal or alkaline earth metal, with theproviso that each A is identical in the same compound.
 2. A compoundselected from those of the formula: ##STR11##
 3. A compound according toclaim 2,4-hydroxy-5-{m-[3-(α,α,α-trifluoro-m-tolyl)ureido]benzamido}-2,7-naphthalenedisulfonicacid, disodium salt.
 4. A compound according to claim 2,4-{m-{3-[p(fluorosulfonyl)phenyl]ureido}benzamido}-5-hydroxy-2,7-naphthalenedisulfonicacid, disodium salt.